Project Summary: Uncovering a new role of nucleosomes in gene regulation Transcriptional factors (TFs) and nucleosomes are two major determinants for gene regulation in eukaryotic cells. Traditionally, TFs and nucleosomes are considered to be mutually exclusive. Recent studies have revealed a group of TFs known as pioneer TFs, including the tumor suppressor p53, which are able to bind nucleosomal DNA without disrupting the overall nucleosome structure. We have established structural rules to account for the accessibility of p53 nucleosomal binding sites, in which the rotational setting of a p53 site on a nucleosome determines its own accessibility. We have also established a cell system using the colon cancer HCT116 cells, in which p53 induces its target genes in different time frames. However, it remains unclear whether the observed temporal regulation of p53 target genes is related to the accessibility of p53 binding sites on nucleosomes. This proposal will use this cell system to discover new mechanisms of nucleosome-driven transcriptional regulation by p53. In Aim 1, we will conduct time-course experiments to examine the accessibility of p53 nucleosomal binding sites associated with early- or late-induced genes in response to DNA damage. In Aim 2, we will study the chromatin organization of p53 binding sites residing in retrotransposons including Alu and LTR/ERV elements, and uncover the biological significance of these binding events. The proposed research seeks to understand previously unappreciated regulatory roles of nucleosomes in the context of chromatin. It will enhance our understanding about how p53 and other pioneer TFs recognize their binding sites on nucleosomes and initiate regulatory events prior to gene activation.